Functionalized stilbene derivatives as improved vascular targeting agents

ABSTRACT

Novel stilbenoid compounds and their prodrug forms are disclosed, which serve as potent vascular targeting agents useful for the treatment of solid tumor cancers and other diseases associated with unwanted neovascularization. The novel stilbenoid compounds are tubulin-binding stilbenoid analogs structurally related to combretastatin A-1 and combretastatin A-4. The prodrug forms serve as potent vascular targeting agents (VTAs) useful for the treatment of solid tumor cancers and diseases associated with retinal neovascularization.

RELATED INVENTION

This application claims priority benefit under 35 U.S.C. § 119(e) ofU.S. provisional patent application Ser. No. 60/337,348 filed on Oct.26, 2001, and is a divisional of U.S. patent application Ser. No.10/281,528 filed Oct. 28, 2002 now U.S. Pat. No. 6,919,324. The entirecontents of each application are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to new stilbenoid compounds and theirprodrug forms, which serve as potent vascular targeting agents usefulfor the treatment of solid tumor cancers and other diseases associatedwith unwanted neovascularization.

More particularly, the present invention relates to tubulin-bindingstilbenoid analogs structurally related to combretastatin A-1 andcombretastatin A-4.

BACKGROUND OF INVENTION

The discovery of the natural products collectively known as thecombretastatins from a willow tree (Combretum caffrum) in South Africaushered in a new era in the development of antimitotic agents whichinhibit the assembly of tubulin into microtubules. Combretastatin A-4(CA-4) and combretastatin A-1 (CA-1), which have the structures:

are especially potent in terms of in vitro cytotoxicity against humancancer cell lines and in their ability to inhibit the assembly oftubulin into microtubules through a direct interaction at the colchicinebinding site on β-tubulin.

It is interesting and instructive to note that while both CA-4 and CA-1are potent inhibitors of tubulin assembly and are strongly cytotoxicagainst human cancer cell lines (Table 1), both of these in vitro assayssuggest that CA-4 is more active biologically than CA-1.

TABLE 1 In Vitro Evaluation of Combretastatins and CombretastatinProdrugs Inhibition of Tubulin MTT Cytotoxicity MTT CytotoxicityPolymerization (IC₅₀) (IC₅₀) at 1 hour (IC₅₀) at 5 hours CA-4 1-2 uM 0.1uM 0.05 uM CA-1 2-4 uM  10 uM 0.05 uM CA-4P >40 uM 0.8 uM 0.002 uM CA-1P >40 uM 3.2 uM 0.0046 uM 

However, when both of these analogs are converted to their correspondingprodrug forms (CA-4P and CA-1P accordingly) and evaluated in vivo interms of tumor vascular shut-down (FIG. 1) and tumor growth delay (FIG.2), then it is apparent that CA-1P is eight to ten-fold more active thanCA-4P in SCID mice. CA4P and CA1P have the structures:

In the case of CA-1P, the most probable biological mode of actionultimately appears to be an enzymatic cleavage by non-specific alkalinephosphatase (or a related enzyme) converting CA-1P (which is not activewith tubulin) to the parent CA-1 (which is active with tubulin). CA-1inhibits the assembly of cytoskeletal tubulin into microtubulesresulting in a morphological change in the endothelial cells lining themicrovessels of tumors. This morphological change causes the endothelialcells to “round-up” which results in an inability of the microvessels tosustain blood flow. Blood clotting and other events ensue whichultimately result in death of the surrounding tumor tissue. Healthytissues are, for the most part, not affected even though the compound isadministered systemically. Several possibilities exist for thisselectivity including (but not limited to): (a) the possibility thatthere is enhanced activity or expression of nonspecific alkalinephosphatase in the micro-environment of the endothelial cells lining thetumor microvessels; (b) potential differences in the tubulin itselfbetween mature healthy cells and immature, rapidly proliferatingendothelial cells in the tumor microvessels which cause enhanceddisruption of the tubulin assembly/disassembly process in the tumormicroenvironment; (c) tumor cells are known to have “leaky” vessels andit is possible that some of the improved tumor growth delay is due tothe compound (as parent drug or prodrug) leaving the blood vessels andentering the cytosol around the tumor where it can form a “supply pool”which ultimately enters the tumor cell itself and (as the parentcompound) functions as an antimitotic agent inhibiting cellular divisionduring metaphase of the cell cycle. The enhanced (10 fold) activity invivo of CA-1P may be due, in part, to the pharmacokinetics associatedwith the cleavage of both of the phosphate groups (perhaps one cleavesmore rapidly than the other) and the subsequent interaction of theparent diphenol (or perhaps one, or both, of themonophenols/monophosphates) with tubulin.

It has therefore been an object of the studies which led to the presentinvention to demonstrate and confirm that the enhanced activity of CA-1Pmay not be due entirely to the substitution pattern in the B-ring of2,3-diphosphate salt, but rather may be due to a change inpharmacokinetics associated with a Z-stilbenoid compound whichincorporates a 3,4,5-trimethoxyphenyl motif in the A-ring, and a4′-methoxy, 3′-O-Phosphate, along with the incorporation of anadditional group (with either an electronic or steric bias) at C-2′,C-5′, or C-6′. Compounds of this basic structural pattern maydemonstrate good bioavailability and favorable pharmacokinetics whichresult in an improved interaction with tubulin and enhanced efficacy asVTAs. It should be readily apparent to anyone skilled in the art thatalthough the new compounds described herein have a trimethoxyarylsubstitution pattern in the A-ring, it is a logical extension to varythe positions of these methoxy groups in the C-2, C-3, C-4, C-5, and C-6positions. Substitution patterns of this type may also result incompounds active as VTAs.

A variety of studies have suggested that the 3,4,5-trimethoxysubstitution pattern on the A-ring and the 4-methoxy moiety on theB-ring are important structural features of the pharmacophore for thesestilbenoid analogs (FIG. 3). Accordingly, the inventors have maintainedthese functionalities in most of the new molecules and have includedfurther substitution patterns around the B-ring. The present inventionand the compounds which are a part thereof is not limited in thisrespect, however, and substitution by other than a 4-methoxy moiety onthe B-ring is contemplated. It is the contention of the presentinventors that the improved in vivo activity of CA-1P (as related toCA-4P) is not due solely to the presence of a diphosphate moiety, butrather may have a strong tie to the pharmacokinetics of this compoundincluding the enzymatic cleavage of the phosphate group (presumably bynonspecific alkaline phosphatase), subsequent inhibition of tubulinassembly resulting in morphological changes (rounding-up) of theimmature endothelial cells lining the microvessels of tumors, and theresulting inability of these microvessels to sustain blood flow.Additional pharmacokinetic parameters such as reversibility of tubulinbinding and perhaps incorporation of the parent CA-1 in the cytosolicfluid around the tumor cell itself may also play key biological roles.

SUMMARY OF THE INVENTION

The present invention relates to novel stilbene compounds and moreparticularly to tubulin-binding stilbenoid analogs structurally relatedto combretastatin A-1 and A-4. The synthesis of these new compounds isdisclosed herein, together with experiments that demonstrate theiractivity in vitro and in vivo.

In a first aspect the present invention provides a novel stilbenecompound represented by the structure:

wherein:

-   -   R₁, R₄ and R₅ is independently H, OH, lower alkoxy, NH₂, NO₂,        N₃, NH-R₆, halogen, a phosphate ester salt moiety of the general        formula (—O—P(O)(O⁻M⁺)₂, wherein M is a metal cation or salt        such as Na, K and Li, or —OPO₃R₇R₈;    -   R₂ is H, OH, lower alkoxy, NH₂, NO₂, NH-R₆, or phosphate ester        salt moiety of the general formula (—O—P(O)(O⁻M⁺)₂, wherein M is        a metal cation or salt such as Na, K and Li; or —OPO₃R₇R₈,        wherein NH₂ or OH may cyclize with R₁;    -   R₃ is H, lower alkoxy, or phosphate ester salt moiety of the        general formula (—O—P(O)(O⁻M⁺)₂, wherein M is a metal cation or        salt such as Na, K and Li or —OPO₃R₇R₈;    -   R₆ is an amino acid acylamino group; and    -   R₇ and R₈ is independently lower alkyl, cycloalkyl, aryl or an        ammonium salt (NH₄ ⁺).

In a second aspect the present invention provides a novel stilbenecompound represented by the structure:

wherein:

R₁ is a phosphate ester salt moiety of the general formula(—P(O)(O⁻M⁺)₂, wherein M is a metal cation or salt such as Na, K and Li,—PO₃R₂R₃, or an alkyl sulfonate;

R₂ is an alkyl group or an ammonium salt (NH₄ ⁺); and

R₃ is an alkyl group or a cycloalkyl.

The compounds of formulas I and II as well as analogs thereof, arevascular targeting agents (VTAs) useful for the treatment of solid tumorcancers and diseases associated with unwanted neovascularization such asretinal neovascularization and restenosis, as well as other conditionsof nonmalignant neovascularization. More specifically, the compounds offormula I and II are useful in the treatment of a variety of cancers,including (but not limited to) the following:

-   -   carcinoma, including that of the bladder, breast, colon, kidney,        liver, lung, including small cell lung cancer, esophagus, gall        bladder, ovary, pancreas, stomach, cervix, thyroid, prostate,        and skin, including squamous cell carcinoma;    -   hematopoietic tumors of lymphoid lineage, including leukemia,        acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell        lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins        lymphoma, hairy cell lymphoma and Burkett's lymphoma;    -   hematopoietic tumors of myeloid lineage, including acute and        chronic myelogenous leukemias, myelodysplastic syndrome and        promyelocytic leukemia;    -   tumors of mesenchymal origin, including fibrosarcoma and        rhabdomyosarcoma;    -   tumors of the central and peripheral nervous system, including        astrocytoma, neuroblastoma, glioma and schwannomas; and    -   other tumors, including melanoma, seminoma, teratocarcinoma,        osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid        follicular cancer, anaplastic thyroid cancer and Kaposi's        sarcoma.

It is thus an object of the present invention to provide a method toreduce or prevent retinal and corneal neovascularization via treatmentwith a drug that inhibits the assembly of tubulin into microtubules andwhich are potent vascular targeting agents.

It is also a further object of this invention is to provide a method toreduce or prevent the development of atherosclerosis or restenosis bytreatment with a drug which inhibits the assembly of tubulin intomicrotubules and which are potent vascular targeting agents.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification and the appended claims, the singular forms also includethe plural unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. All patents and publications cited in thisspecification are incorporated herein by reference.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 is a comparison of the effects of CA-4P and CA-1P on tumor bloodflow over time. The vascular shutdown capacity of each compound wasmeasured in SCID mice implanted subcutaneously with a murinehemangioendothelioma (MHEC5-T) tumor (n=3). In each case, 100 mg/kg ofdrug was injected as a single dose, and fluorescent beads were addedthrough a tail vein 3 minutes prior to sacrifice. Blood flow reductionwas quantified by fluorescence microscopy and expressed as a percentageof blood flow observed in those animals treated with a saline control;

FIG. 2 is a comparison of the anti tumor growth activity of CA1-P andCA-4P versus control in a nude mouse model of human breast carcinoma.Mice (n=3) were treated once daily (3.2, 6.3, 12.5, or 25 mg/kg) in thefirst five days of the experiment. Some of the tumors were retreated foranother 5 days beginning on day 42 of the experiment; and

FIG. 3 is depiction of the salient structural-activity relationship(SAR) features of the stilbenoid VTA pharmacophore. These features areretained in the molecular structure of CA-4 and are important foroptimal tubulin-binding and vascular shutdown activity.

DETAILED DESCRIPTION OF THE INVENTION

As defined herein, the present invention provides compounds of formula Iand II and analogs and prodrugs thereof, pharmaceutical compositionsemploying such compounds and methods of using such compounds.

Listed below are definitions of various terms used to describe thecompounds of the instant invention. These definitions apply to the termsas they are used throughout the specification (unless they are otherwiselimited in specific instances) either individually or as part of alarger group.

It should be noted that any heteroatom with unsatisfied valences isassumed to have the hyrdrogen atom to satisfy the valences.

The term alkyl group when used alone or in combination with othergroups, are lower alkyl containing from 1 to 8 carbon atoms and may bestraight chained or branched. An alkyl group is an optionallysubstituted straight, branched or cyclic saturated hydrocarbon group.When substituted, alkyl groups may be substituted with up to foursubstituent groups, R as defined, at any available point of attachment.When the alkyl group is said to be substituted with an alkyl group, thisis used interchangeably with “branched alkyl group”. Exemplaryunsubstituted such groups include methyl, ethyl, propyl, isopropyl,n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl,dodecyl, and the like. Exemplary substituents may include but are notlimited to one or more of the following groups: halo (such as F, Cl, Br,I), haloalkyl (such as CCl₃ or CF₃), alkoxy, alkylthio, hydroxy, carboxy(—COOH), alkyloxycarbonyl (—C(O)R), alkylcarbonyloxy (—OCOR), amino(—NH₂), carbamoyl (—NHCOOR— or —OCONHR—), urea (—NHCONHR—) or thiol(—SH). Alkyl groups as defined may also comprise one or more carbon tocarbon double bonds or one or more carbon to carbon triple bonds.

The preferred alkyl groups contain 1-8 carbon atoms; more preferredalkyl groups contain 1-6 carbon atoms. Alkylene as used herein refers toa bridging alkyl group of the formula C_(n)H_(2n). Examples include CH₂,—CH₂CH₂—, —CH₂ CH₂CH₂— and the like.

As used herein the term “cycloalkyl” is a species of alkyl containingfrom 3 to 15 carbon atoms, without alternating or resonating doublebonds between carbon atoms. It may contain from 1 to 4 rings. Exemplaryunsubstituted such groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, adamantyl, etc. Exemplary substituents include one or moreof the following groups: halogen, alkyl, alkoxy, alkyl hydroxy, amino,nitro, cyano, thiol and/or alkylthio.

As used herein, the term “aryl” refers to groups with aromaticity,including 5- and 6-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, as well as multicyclic systems with atleast one aromatic ring. Examples of aryl groups include benzene,phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like. The aromatic ring can besubstituted at one or more ring positions with such substituents asdescribed above, as for example, halogen, hydroxyl, alkoxy, etc.

As used herein, the term “lower alkoxy” refers to —O-alkyl groups,wherein alkyl is as defined hereinabove. The alkoxy group is bonded tothe main chain, aryl or heteroaryl group through the oxygen bridge. Thealkoxy group may be straight chained or branched; although thestraight-chain is preferred. Examples include methoxy, ethyloxy,propoxy, butyloxy, t-butyloxy, i-propoxy, and the like. Preferred alkoxygroups contain 1-4 carbon atoms, especially preferred alkoxy groupscontain 1-3 carbon atoms. The most preferred alkoxy group is methoxy.

As used herein, the term “halogen” or “halo” refers to chlorine,bromine, fluorine or iodine.

As used herein, the term “lower alkylamino” refers to a group whereinone alkyl group is bonded to an amino nitrogen, i.e., NH(alkyl). The NHis the bridge connecting the alkyl group to the aryl or heteroaryl.Examples include NHMe, NHEt, NHPr, and the like.

The amino acid acyl group in the amino acid acylamino group is an acylgroup derived from the amino acid. The amino acids may be enumerated byα-amino acids, β-amino acids and γ-amino acids. Examples of preferredamino acids include glycine, alanine, leucine, serine, lysine, glutamicacid, asparatic acid, threonine, valine, isoleucine, ornithine,glutamine, asparagines, tyrosine, phenylalanine, cysteine, methionine,arginine, β-alanine, tryptophan, proline, histidine, etc. The preferredamino acid is serine.

As used herein, the term “prodrug” refers to a precursor form of thedrug which is metabolically converted in vivo to produce the activedrug. Thus, for example, combretastatin A-4 phosphate prodrug salts orcombretastatin A-1 phosphate prodrug salts administered to an animal inaccordance with the present invention undergo metabolic activation andregenerate combretastatin A-4 or combretastatin A-1 in vivo, e.g.,following dissociation and exposure to endogenous non-specificphosphatases in the body. A phosphate prodrug salt or phosphate estersalt moiety as used interchangeably herein, include those with aphosphate ester salt moiety (—OP(O)(O⁻M⁺)₂) or one phosphate triestermoiety (—OP(O)(OR)₂) or one phosphate diester moiety (—OP(O)(OR)(O⁻M⁺)where M is a salt and R is chosen to be any appropriate alkyl orbranched alkyl substituent (the two R groups may be the same alkyl groupor may be mixed), or benzyl, or aryl groups. The salt M isadvantageously Na, K and Li, but the invention is not limited in thisrespect. The phosphate ester salt moiety may also include(—OP(O)(O-alkyl)₂ or (—OP(O)(O—NH₄ ⁺)₂).

As used herein, the term “salt” is a pharmaceutically acceptable saltand can include acid addition salts including hydrochlorides,hydrobromides, phosphates, sulphates, hydrogen sulphates,alkylsulphonates, arylsulphonates, acetates, benzoates, citrates,maleates, fumarates, succinates, lactates, and tartrates; alkali metalcations such as Na, K, Li, alkali earth metal salts such as Mg or Ca ororganic amine salts such as those disclosed in PCT InternationalApplication Nos. WO02/22626 or WO00/48606.

All stereoisomers of the compounds of the instant invention arecontemplated, either in admixture or in pure or substantially pure form.The definition of the compounds according to the invention embraces allpossible stereoisomers and their mixtures. It very particularly embracesthe racemic forms and the isolated optical isomers having the specifiedactivity. The racemic forms can be resolved by physical methods, suchas, for example, fractional crystallization, separation orcrystallization of diastereomeric derivatives or separation by chiralcolumn chromatography. The individual optical isomers can be obtainedfrom the racemates by conventional methods, such as, for example, saltformation with an optically active acid followed by crystallization.

The present invention also relates to the administration of a vasculartargeting agent, particularly a tubulin binding agent having thechemical structures disclosed herein, for treating malignant ornon-malignant vascular proliferative disorders.

Tubulin binding agents inhibit tubulin assembly by binding totubulin-binding cofactors or cofactor-tubulin complexes in a cell duringmitosis and prevent the division and thus proliferation of the cell.Tubulin binding agents comprise a broad class of compounds which inhibittubulin polymerization, and which generally function as tumor selectivevascular targeting agents useful for cancer chemotherapy, as well as forother non-cancer applications such as ocular disease and restenosis.

Vascular Targeting Agents, also known as Vascular Damaging Agents, are anovel class of antineoplastic drugs which attack solid tumors byselectively occluding, disrupting, or destroying the existingvasculature formed by angiogenesis. The cytotoxic mechanism of VTAaction is quite divorced from that of anti-angiogenic agents. A singledose of VTA can cause a rapid and irreversible tumor vascular shutdownof existing tumor vasculature, leading eventually to tumor necrosis byinduction of hypoxia and nutrient depletion. Other agents have beenknown to disrupt tumor vasculature but differ in that they also manifestsubstantial normal tissue toxicity at their maximum tolerated dose. Incontrast, genuine VTAs retain their vascular shutdown activity at afraction of their maximum tolerated dose.

In one embodiment, the present invention is directed to theadministration of a vascular targeting agent (“VTA”), particularly atubulin binding agent, for the treatment of malignant or non-malignantvascular proliferative disorders in ocular tissue.

Neovascularization of ocular tissue is a pathogenic conditioncharacterized by vascular proliferation and occurs in a variety ofocular diseases with varying degrees of vision failure. Theadministration of a VTA for the pharmacological control of theneovascularization associated with non-malignant vascular proliferativedisorders such as wet macular degeneration, proliferative diabeticretinopathy or retinopathy of prematurity would potentially benefitpatients for which few therapeutic options are available. In anotherembodiment, the invention provides the administration of a VTA for thepharmacological control of neovascularization associated with malignantvascular proliferative disorders such as ocular tumors.

The blood-retinal barrier (BRB) is composed of specializednonfenestrated tightly-joined endothelial cells that form a transportbarrier for certain substances between the retinal capillaries and theretinal tissue. The nascent vessels of the cornea and retina associatedwith the retinopathies are aberrant, much like the vessels associatedwith solid tumors. Tubulin binding agents, inhibitors of tubulinpolymerization and vascular targeting agents, may be able to attack theaberrant vessels because these vessels do not share architecturalsimilarities with the blood retinal barrier. Tubulin binding agents mayhalt the progression of the disease much like they do with atumor-vasculature. Local (non-systemic) delivery of tubulin bindingagents to the eye can be achieved using intravitreal injection,sub-Tenon's injection, ophthalmic drops iontophoresis, and implantsand/or inserts. Systemic administration may be accomplished byadministration of the tubulin binding agents into the bloodstream at asite which is separated by a measurable distance from the diseased oraffected organ or tissue, in this case they eye. Preferred modes ofsystemic administration include parenteral or oral administration.

The compounds of the present invention may are also contemplated for usein the treatment of vascular disease, particularly atheroscleorsis andrestenosis. Atherosclerosis is the most common form of vascular diseaseand leads to insufficient blood supply to critical body organs,resulting in heart attack, stroke, and kidney failure. Additionally,atherosclerosis causes major complications in those suffering fromhypertension and diabetes, as well as tobacco smokers. Atherosclerosisis a form of chronic vascular injury in which some of the normalvascular smooth muscle cells (“VSMC”) in the artery wall, whichordinarily control vascular tone regulating blood flow, change theirnature and develop “cancer-like” behavior. These VSMC become abnormallyproliferative, secreting substances (growth factors, tissue-degradationenzymes and other proteins) which enable them to invade and spread intothe inner vessel lining, blocking blood flow and making that vesselabnormally susceptible to being completely blocked by local bloodclotting, resulting in the death of the tissue served by that artery.

Restenosis, the recurrence of stenosis or artery stricture aftercorrective surgery, is an accelerated form of atherosclerosis. Recentevidence has supported a unifying hypothesis of vascular injury in whichcoronary artery restenosis along with coronary vein graft and cardiacallograft atherosclerosis can be considered to represent a muchaccelerated form of the same pathogenic process that results inspontaneous atherosclerosis (Ip, J. H., et al., (1990) J Am CollCardiol, 15:1667-1687; Muller, D. W. M., et al., (1992) J Am CollCardiol, 19:418-432). Restenosis is due to a complex series offibroproliferative responses to vascular injury involving potentgrowth-regulatory molecules, including platelet-derived growth factor(PDGF) and basic fibroblast growth factor (bFGF), also common to thelater stages in atherosclerotic lesions, resulting in vascular smoothmuscle cell proliferation, migration and neointimal accumulation.

Restenosis occurs after coronary artery bypass surgery (CAB),endarterectomy, and heart transplantation, and particularly after heartballoon angioplasty, atherectomy, laser ablation or endovascularstenting (in each of which one-third of patients redevelopartery-blockage (restenosis) by 6 months), and is responsible forrecurrence of symptoms (or death), often requiring repeatrevascularization surgery. Despite over a decade of research andsignificant improvements in the primary success rate of the variousmedical and surgical treatments of atherosclerotic disease, includingangioplasty, bypass grafting and endarterectomy, secondary failure dueto late restenosis continues to occur in 30-50% of patients (Ross, R.(1993) Nature, 362:801-809).

The most effective way to prevent this disease is at the cellular level,as opposed to repeated revascularization surgery which can carry asignificant risk of complications or death, consumes time and money, andis inconvenient to the patient.

Microtubules, cellular organelles present in all eukaryotic cells, arerequired for healthy, normal cellular activities. They are an essentialcomponent of the mitotic spindle needed for cell division, and arerequired for maintaining cell shape and other cellular activities suchas motility, anchorage, transport between cellular organelles,extracellular secretary processes (Dustin, P. (1980) Sci. Am., 243:66-76), as well as modulating the interactions of growth factors withcell surface receptors, and intracellular signal transduction.Furthermore, microtubules play a critical regulatory role in cellreplication as both the c-mos oncogene and CDC-2-kinase, which regulateentry into mitosis, bind to and phosphorylate tubulin (Verde, F. et al.(1990) Nature, 343:233-238), and both the product of the tumorsuppressor gene, p53, and the T-antigen of SV-40 bind tubulin in aternary complex (Maxwell, S. A. et al. (1991) Cell Growth Differen.,2:115-127). Microtubules are not static, but are in dynamic equilibriumwith their soluble protein subunits, the α- and β-tubulin heterodimers.Assembly under physiologic conditions requires guanosine triphosphate(GTP) and certain microtubule associated and organizing proteins ascofactors; on the other hand, high calcium and cold temperature causedepolymerization.

Interference with this normal equilibrium between the microtubule andits subunits would therefore be expected to disrupt cell division andmotility, as well as other activities dependent on microtubules. Thisstrategy has been used with significant success in the treatment ofcertain malignancies. Indeed, antimicrotubule agents such as colchicineand the vinca alkaloids are among the most important anticancer drugs.These antimicrotubule agents, which promote microtubule disassembly,play principal roles in the chemotherapy of most curable neoplasms,including acute lymphocytic leukemia, Hodgkin's and non-Hodgkin'sLymphomas, and germ cell tumors, as well as in the palliative treatmentof many other cancers.

Taxol® (paclitaxel) has been shown to be an effective antimicrotubuleagent. Unlike other antimicrotubules such as colchicine and the vincaalkaloids which promote microtubule disassembly, taxol acts by promotingthe formation of unusually stable microtubules, inhibiting the normaldynamic reorganization of the microtubule network required for mitosisand cell proliferation (Schiff, P. B., et al. (1979) Nature 277: 665;Schiff, P. B., et al. (1981) Biochemistry 20: 3247). In the presence oftaxol, the concentration of tubulin required for polymerization issignificantly lowered; microtubule assembly occurs without GTP and atlow temperatures, and the microtubules formed are more stable todepolymerization by dilution, calcium, cold, and inhibitory drugs. Taxolwill reversibly bind to polymerized tubulin, and other tubulin-bindingdrugs will still bind to tubulin even in the presence of taxol.

Taxol is, however, highly insoluble and severe allergic reactions havebeen observed following administration of taxol. Furthermore, cardiacarrhythmias are associated with taxol administration, and like allergicreactions, their incidence is affected by the dosage and rate of taxoladministration.

Although others have investigated the use of the antimicrotubule agentcolchicine in preventing restenosis, opposite conclusions have beenreported (See Currier, et al., “Colchicine Inhibits Restenosis AfterIliac Angioplasty In The Atherosclerotic Rabbit” (1989) Circ., 80:II-66;O'Keefe, et al., “Ineffectiveness Of Colchicine For The Prevention OfRestenosis After Coronary Angioplasty” (1992) J. Am. Coll. Cardiol.,19:1597-1600). The art, however, fails to suggest the use of a vasculartargeting agent such as CA-1 or CA-4 in preventing or reducingrestenosis. Thus, the method of the present invention is to prevent orreduce the development of atherosclerosis or restenosis using a vasculartargeting agent such as CA-1 or CA-4 or their analogs, as well asprodugs of these compounds. This microtubule stabilizing mechanism ofatherosclerosis or restenosis prevention is supported by the analogousresults in experiments on cellular proliferation and migration usingtaxol and H₂O (deuterium oxide), which exert comparable microtubuleeffects via different underlying mechanisms.

Pharmaceutical compositions of the invention are formulated to becompatible with its intended route of administration. As with the use ofother chemotherapeutic drugs, the individual patient will be monitoredin a manner deemed appropriate by the treating physician. Dosages canalso be reduced if severe neutropenia or severe peripheral neuropathyoccurs, or if a grade 2 or higher level of mucositis is observed, usingthe Common Toxicity Criteria of the National Cancer Institute.

Pharmaceutical compositions for ophthalmic topical administration mayinclude ophthalmic solutions, ophthalmic gels, sprays, ointments,perfusion and inserts. A topically delivered formulation of tubulinbinding agent should remain stable for a period of time long enough toattain the desired therapeutic effects. In addition the agent mustpenetrate the surface structures of the eye and accumulate insignificant quantities at the site of the disease. Additionally, atopically delivered agent should not cause an excessive amount of localtoxicity.

Ophthalmic solutions in the form of eye drops generally consist ofaqueous media. In order to accommodate wide ranges of drugs which havevarious degrees of polarity, buffers, organic carriers, inorganiccarriers, emulsifiers, wetting agents, etc. can be added.Pharmaceutically acceptable buffers for ophthalmic topical formulationsinclude phosphate, borate, acetate and glucoronate buffers, amongstothers. Drug carriers may include water, water mixture of loweralkanols, vegetable oils, polyalkylene glycols, petroleum based jelly,ethylcellulose, ethyl oleate, carboxymethylcellulose,polyvinylpyrrolidone, and isoproplyl myristrate. Ophthalmic spraysgenerally produce the same results as eye drops and can be formulated ina similar manner. Some ophthalmic drugs have poor penetrability acrossocular barriers and are not administrable as drops or spray. Ointmentsmay thus be used to prolong contact time and increase the amount of drugabsorbed. Continuous and constant perfusion of the eye with drugsolutions can be achieved by placing polyethylene tubing in theconjunctival sac. The flow rate of the perfusate is adjustable via aminipump system to produce continuous irrigation of the eye. Inserts aresimilar to soft contact lens positioned on the cornea, except thatinserts are generally placed in the upper cul-de-sac or, lessfrequently, in the lower conjunctival sac rather than attached to theopen cornea. Inserts are generally made of biologically solublematerials which dissolve in lacrimal fluid or disintegrate whilereleasing the drug.

The compositions of the present invention may also be formulated forsystemic administration. Examples of systemic routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transmucosal, and rectal administration. Solutionsor suspensions used for parenteral or subcutaneous application caninclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a vascular targeting agent) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The compound of the present invention may also prove useful in thetreatment of coronary artery disease by serving as antimitotic agentscoated (or conjugated) onto stents to prevent the recurring problem ofrestenosis after angioplasty.

In addition to the vascular targeting agents described above, theinvention also includes the use of pharmaceutical compositions andformulations comprising a vascular targeting agent in association with apharmaceutically acceptable carrier, diluent, or excipient, such as forexample, but not limited to, water, glucose, lactose, hydroxypropylmethylcellulose, as well as other pharmaceutically acceptable carriers,diluents or excipients generally known in the art.

As used herein, terms “pharmacologically effective amount”,“pharmaceutically effective dosage” or “therapeutically effectiveamount” mean that amount of a drug or pharmaceutical agent that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by a researcher or clinician.

It is intended that the systemic and non-systemic administration of VTAsand tubulin binding agents in accordance with the present invention willbe formulated for administration to mammals, particularly humans.However, the invention is not limited in this respect and formulationsmay be prepared according to veterinary guidelines for administration toanimals as well.

The vast majority of the compounds described herein can be preparedsynthetically through a Wittig reaction between an appropriatelysubstituted aldehyde and an appropriately substituted phosphorous ylide.The aldehyde portion and ylide portion can be readily switched as wellto allow for the judicious incorporation of the requisite functionalgroups within the target stilbenoids (see Scheme 1 and 2 for generalsynthetic protocols).

A wide variety of functionalized stilbenoid compounds have been preparedutilizing the general synthetic approach outlined in Schemes 1 and 2. Ineach case, the starting materials can either be purchased (AldrichChemical Co., and/or Acros (Fisher Scientific), etc.) or prepared in oneor two steps by routes described in the literature. It is important tonote that each of these preferred compounds contains the3,4,5-trimethoxyphenyl motif in the A-ring of the stilbenoid along withadditional functionalization in terms of groups with steric and/orelectronic bias at the remaining positions in the B-ring. The preferredstereochemical configuration is Z, however it should be readily apparentto anyone skilled in the art that the corresponding E-isomers will bereadily obtained and certain of these E-isomers may have activity asVTAs. In each case, the free phenolic moiety can be readily converted toits corresponding phosphate prodrug entity as exemplified for one of thecompounds in Scheme 3. Where more than one free phenolic moiety ispresent, a mixture of partially phosphorylated compounds can be producedby using limiting amounts of the reagents in Scheme 3

A logical developmental extension of the phenolic stilbenoid analogs isthe replacement of the phenol moiety with an amine functionality. Thisamine can be further modified to form an amide bond to an amino acidresidue and function biologically as a prodrug. The parent free aminestill functions biologically through a binding interaction with tubulin,while the amide prodrug linkage (serinamide, for example) serves as aprodrug construct. This concept has been successfully developed byAjinomoto Pharmaceuticals Co., Inc. through the preparation of the3′-amino analog of CA-4 and its corresponding serinamide. Utilizingsimilar synthetic methodology as employed by Ajinomoto Inc. (Scheme 4),we have prepared a variety of amine functionalized stilbenoid compoundsand their corresponding serinamide congeners.

It is important to that certain of these compounds can contain both aserinamide and a phosphate salt or ester. In addition, certain of thesecompounds can contain a phosphate salt judiciously bridged between aphenol and an amine functionality. It is important to note that althoughserine may be the preferred amino acid to use in these prodrugformulations, other amino acids may be utilized as well. All of thesetransformations to prodrugs can be achieved by the methods describedherein as well as through the use of other standard syntheticmethodologies which should be obvious to anyone skilled in the art.

In an effort to improve the bioavailabilty and pharmacokinetics ofstilbenoid derivatives as VTAs, a CA-4P dimer has been prepared (Scheme5).

In an analogous manner, a variety of other stilbenoid dimers areanticipated which could be readily prepared by a very similar syntheticstrategy to that illustrated in Scheme III. In addition, other saltcounter ions such as lithium, potassium, etc. may be employed withpresumably equivalent efficacy.

The potential biological advantage of the dimers is based on thefollowing strategy:

-   -   A) The dimers may prove to be poorer substrates for enzymatic        cleavage by nonspecific alkaline phosphatase or other enzyme    -   B) By slowing down the cleavage of the phosphate (prodrug        portion of the molecule), the pharmacokinetics may be altered in        a favorable fashion resulting in improved function in terms of        vascular targeting    -   C) The dimers are especially attractive due to the fact that        enzymatic cleavage delivers two molecules of the biologically        potent stilbenoid VTA.

A variety of triester and diester phosphates have been prepared in orderto address the issue of improved pharmacokinetics leading to enhancedvascular targeting capability. Several phosphate diester CA4 prodrugshave been prepared based on the promising biological activity displayedby the parent phosphate monoester prodrug, CA4P. A general synthesis forthese compounds is detailed in Scheme 6. It should be obvious to anyoneskilled in the art that an enormous variety of diesters derived from CA4(and analogously, derived from other phenolic combretastatins as well asdiols such as CA1) can readily be prepared using the methodologiesdescribed herein.

The design premise for these new compounds is similar to that outlinedand developed for the stilbenoid dimers (previously described). Asimilar synthetic strategy was employed for the synthesis of theseligands (see Schemes 6 and 7 for representative examples). The compoundsclearly demonstrate reduced cytotoxicity (compared to CA-4) which doessuggest that they may be poorer substrates for enzymatic cleavage of thephosphate moiety which may prove advantageous for improved VTAs in termsof enhanced in vivo pharmacokinetic profiles.

The invention is further defined by reference to the following examplesand preparations which describe the manner and process of making andusing the invention and are illustrative rather than limiting. It willbe apparent to those skilled in the art that many modifications, both tothe materials and methods, may be practiced without departing from thepurpose and interest of the invention.

EXAMPLES Example 1 2′Hydroxy-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-26A)

A. Preparation of 3,4,5-trimethoxybenzyltriphenylphosphonium bromide

To a well-stirred solution of CBr₄ (5.10 g, 15.4 mmol) in acetone (80mL) at 0° C. under N₂, 3,4,5-trimethoxybenzyl alcohol (2.23 g, 11.3mmol) and triphenylphosphine (4.00 g, 15.3 mmol) were added. After 12hours, the mixture was filtered through Celite and the solvent removedunder reduced pressure to yield benzyl bromide as a brown oil. This oilwas then dissolved in CH₂Cl₂ (50 mL) and PPh₃ (3.25 g, 12.4 mmol) wasadded. The reaction was heated overnight and then ice-cold water wasadded and the product isolated by extraction with CH₂Cl₂. The organicphase was washed with brine and dried over sodium sulfate. Evaporationof the solvent in vacuo resulted in a crude solid, which wasrecrystallized from ethyl alcohol/hexane to afford3,4,5-trimethoxybenzyltriphenyl phosphonium bromide (5.0 g, 85%).

B. Preparation of 2-hydroxy-3-bromo-4-methoxybenzaldehyde

Treatment of 2-hydroxy-4-methoxybenzaldehyde (3.04 g, 20 mmole) withmercuric acetate (1 eq., 20 mmole) in refluxing ethanol (100 ml)containing acetic acid (1% weight percentage) followed by treatment withaq. NaBr gave in 80% yield a mixture of organo-mercusy compounds. Themixture was treated with 1 eq. of Bromine in CHCl₃ containing a smallamount of acetic acid. Purification over silica gel (elution with 30%EtOAc in hexane, afforded 2-hydroxy-3-bromo-4-methoxybenzaldehyde (2.11g, 47.2%)

C. Preparation of 2-(t-butyldimethylsilyl)-3-bromo-4-methoxybenzaldehyde

Diiospropylethylamine (3.0 ml) was added to a stirred solution (underargon) of 2-hydroxy-3-bromo- -4-methoxybenzaldehyde (1.96, 8.5 mmole) inDMF (15 ml) followed by t-butyldimethlsilyl chloride (TBSCl, 1.91 g,12.8 mmole). The reaction mixture was stirred at room temperature for 30min and Ice (20 g) was added to the mixture. The mixture was thenextracted with ether (3×25 ml). The ethereal solution was washed withwater (25 ml) and saturated NaHCO₃ solution (2×15 ml). The solventevaporated to yield 2-(t-butyldimethylsilyl)-3-bromo-4-methoxybenzaldehyde as an oil (2.54 g, 7.06 mmole, 83.1%).

D. Preparation of2′-oxy-(t-butyldimethylsilyl)-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene

Butyllithium (1.5 ml, 2M Hexane, 3 mmole) was added (under argon) to asuspension of 3,4,5-trimethoxybenzyltryphenylphosphonium bromide (1.57g, 3 mmole) in THF (50 ml) at −15° C. The resulting deep reddishsolution was allowed to stir at room temperature for 30 min.2-hydroxy-3-bromo-4-methoxybenzaldehyde (0.991 g, 2.8 mmole) was added,and the reaction mixture was kept stirring for 3 hours. The reactionmixture was diluted with ice-cold H₂O and extracted with ether (3×25ml). The etheral solution was washed with water, and solvent wasevaporated to yield a Z and E mixture of2′-oxy-(t-butyldimethylsilyl)-3′-bromo-3,4,4′,5-tetramethoxystilbene(1.20 g mixture, 2.36 mmole, 78.7%).

E. Preparation of 2′-hydroxy-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene

To a DMF (7 ml) solution containing Z&E mixture of2′-oxy-(t-butyldimethylsilyl)-3′-bromo-3,4,4′,5-tetramethoxystilbene(743 mg 1.46 mmole), KF (84 mg, 1.46 mmole) and HBr (0.17 ml, 1.46mmole) was added. The reaction was monitored by TLC. Another 0.17 ml HBrwas added in the second day. The reaction was kept stirring for 2 days.Water (15 ml) was added to the solution, and the solution was extractedwith ethyl acetate (3×15 ml). The extraction was washed with water,dried with sodium sulfate, and rotavapored. The residue was applied tosilica gel column and eluted with hexane:ethyl acetate (7:3) to afford2′-hydroxy-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene (256 mg 0.64mmole, 43.8%).

H-NMR, (ppm, δ): 6.95, (1H, d, d=11.5 Hz), 6.56(1H, d, J=8.5 Hz),6.52(1H, d, J=11.5 Hz), 6.44(2H, s), 3.80(3H, s), 3.61(3H, s), 3.54(3H,s)

C-NMR, (ppm, δ): 155.00, 153.38, 150.72, 128.40, 126.07, 122.48, 118.59,133.00, 103.93, 103.49, 60.98, 56.44, 56.15.

Example 2 2′-DisodiumPhosphate-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene

A. Preparation of2-O-Bis(benzyl)phosphoryl-3-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene

2′-hydroxy-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene (250 mg, 0.63mmole) was dissolved in acetonitrile (10 ml) in a flask equipped with aseptum, thermometer and argon inlet. After cooling to −25° C., carbontetrachloride (5 eq. 3.15 mmole, 0.6 ml) was added and the solution wasstirred for 5 min. With a syringe, diisopropylethylamine (0.65 ml, 2eq.) was added followed by DMAP(18 mg. 0.3 eq, 0.15 mmole). Slowaddition of dibenzyl phosphite (0.25 ml, 1.26 mmole, 2.0 eq.) was begun1 min later at such a rate the reaction temperature remained below −20°C. After completion of the reaction (in 1 hour by TLC analysis), 0.5MKH₂PO₄ was added (5 ml), and the solution was allowed to warm to roomtemperature and extracted with ethyl acetate (3×20 ml). The combinedsolvent extract was washed with water (25 ml) and saturated NaCl (25ml), then dried. Filtration and removal of solvent gave an oil that waschromatographed on a column of silica gel (hexane:ethyl acetate, 4:1) togive2-O-Bis(benzyl)phosphoryl-3-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene(390 mg, 94.5%) as a clear gum.

B. Preparation of 2′-DisodiumPhosphate-3′-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene

Chlorotrimethylsilane (70 mg, 0.648 mmole, 0.082 ml, 2 eq.) was slowlyadded (with vigorous stirring) to a solution of2-O-Bis(benzyl)phosphoryl-3-bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene(212 mg, 0.324 mmole) and sodium iodide (97.2 mg, 0.648 mmole) in dryacetonitrile (5 ml, in a dry flask under argon). After stirring 20 min,TLC analysis, showed no starting material. Enough water was added todissolve the salts and a straw color was removed by the addition of 10%aq. sodium thiosulfate (5 drops). The solvent was separated and theaqueous phase extracted with ethyl acetate (4×10 ml). The combinedextracted was concentrated in vacuo, and the resulting foam wasdissolved in dry methanol (2 ml). Sodium Methoxide (95%, 34 mg, 0.648mmole) was added in one portion and the solution stirred for 9 hours.The methanol was removed under reduced pressure and the solidrecrystallized from water-ethanol to give a white powder (64 mg, 0.12mmole, 37.0%)

HNMR(ppm, δ): 6.98, (1H, d, d=11.5 Hz), 6.58(1H, d, J=8.5 Hz), 6.50(1H,d, J=11.5 Hz), 6.48(2H, s), 3.83(3H, s), 3.63(3H, s), 3.55(3H, s).PNMR(ppm, δ): −0.36

Example 3 2′,3′-Dinitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-3B)

4-methoxy-2,3-dinitrobenzaldehyde (2.94 mmol) and3,4,5-trimethoxybenzyltriphenyl phosphonium bromide (1.54 g, 2.94 mmol,1.0 equiv) in anhydrous dichloromethane (25 mL) was added NaH (0.424 g,17.67 mmol, 6.0 equiv). The reaction mixture was stirred at roomtemperature for about 7 hours and monitored by TLC. The reaction wasquenched by adding water, the organic layer was separated and theaqueous layer was extracted with dichloromethane (3×25 mL). The combinedorganic layer was washed with brine, dried over Na₂SO₄ and concentratedin vacuo to orange colored slush. To this was added about 15 mL ofdichloromethane and refrigerated overnight. The crude mixture wassubjected to flash chromatography to isolate2′,3′-Dinitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (0.581 g, 1.48 mmol,51%, solid)

¹H NMR (300 MHz, CDCl₃): δ 3.69 (6H, s), δ 3.82 (3H, s), δ 3.95 (3H, s),δ 6.30 (2H, s), δ 6.49 (1H, d, J=11.86), δ 6.77 (1H, d, J=11.84 Hz), δ7.09 (1H, d, J=8.93 Hz), δ 7.36 (1H, d, J=8.9 Hz).

Example 4 2′,3′-Damino-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-3B)

A well-stirred solution of2′,3′-Dinitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (0.422 g, 1.08 mmol) ina mixture of acetone-water (2:1) was heated to 50° C. Then sodiumthiosulfate (1.88 g, 10.81 mmol, 10.0 equiv) was added and the reactionmixture was heated to reflux for 6 hours. The reaction was cooled toroom temperature and water was added. The organic layer was separatedand the aqueous layer was extracted with ethyl acetate (4×25 mL). Thecombined organic layer were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. The mixture was then subjected to preparative TLCto give the 2′,3′-Diamino-3,4,4′,5-tetramethoxy-(Z)-stilbene.

¹H NMR (360 MHz, CDCl₃): δ3.61 (6H, s), δ 3.80 (3H, s), δ 3.82 (3H, s),δ 6.38 (1H, d, J=8.44 Hz), 66.48 (1H, d, J=12.12 Hz), δ 6.49 (2H, s), δ6.52 (1H, d, J=12.06 Hz), δ 6.66 Hz (1H, d, J=8.43 Hz).

Example 5 2′serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-45)

A. Preparation of2′-FMOC-L-serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene

To a well stirred solution of2′-amino-3,4,4′,5-tetramethoxy-(Z)-stilbene (0.114 g, 0.362 mmol) inanhydrous DMF (2 mL) were added DCC (0.101 g, 0.489 mmol), FMOC(Ac)L-serinamide (0.173 g, 0.467 mmol), and HOBt.H₂0 (0.0702 g, 0.520 mmol)at room temperature. After 21.5 h, EtOAc was added and the mixture wasfiltered. The filtrate was washed 5 times with water and twice withbrine and the organic phase was dried over sodium sulfate. Afterevaporation of the solvent the yellow oil was purified by normal-phasepreparative TLC (60% hex-EtOAc) developing twice to afford2′-FMOC-L-serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene (0.1308 g, 54%yield). ¹H NMR (CDCl₃, 300 MHz) 68.13 (br, 1H), 7.91 (s, 1H), 7.77 (d,2H, J=7 Hz), 7.57 (br, 2H), 7.40 (br, 2H), 7.31 (br, 2H), 7.13 (d, 1H,J=8.5), 6.70 (dd, 1H, J=8.5, 2.4 Hz), 6.32 (br, 2H), 6.22 (s, 2H), 5.27(m, 1H), 4.54 (m, 2H), 4.40 (m, 1H), 4.19 (m,1H), 3.80 (s, 3H), 3.72 (s,3H), 3.48 (s, 6H), 1.95 (s, 3H).

B. Preparation of 2′ serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene

2′-FMOC-L-serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene (0.131 g, 0.226mmol) was dissolved in 1.5 mL of dichloromethane and 1.5 mL of MeOH and0.22 mL (0.0176 g, 0.44 mmol) of an aqueous solution of 2N— sodiumhydroxide were added. After the reaction mixture was stirred at roomtemperature for 18 h dichloromethane was added and the organic phase waswashed once with water and twice with brine, dried under sodium sulfateand the solvent evaporated. The resulting oil was purified bynormal-phase preparative TLC (95% CH₂Cl₂—MeOH) to afford 2′serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene (52.9 mg, 67%). ¹H NMR(CDCl₃, 300 MHz) δ 9.65 (s, 1H), 8.02 (d, 1H, J=2.6 Hz), 7.15 (d, 1H,J=8.5 Hz), 6.68 (dd, 1H, J=8.5, 2.6 Hz), 6.60 (d, 1H, J=12.1 Hz), 6.49(d, 1H, J=12 Hz), 3.81 (s, 3H), 3.79 (s, 3H), 3.72 (m, 1H), 3.62 (m,1H), 3.60 (s, 6H), 3.36 (t, 1H, J=5.3 Hz), 1.80 (br, 2H). ¹³C NMR(CDCl₃, 75 MHz) δ 171.8, 159.5, 152.8, 137.7, 135.8, 132.6, 131.8,130.1, 124.3, 119.9, 110.9, 105.8, 105.2, 65.1, 60.9, 56.5, 55.9, 55.5.

Example 6 CA-4P Methyl Ester, Ammonium Salt (Oxi-com-209)

Step A

0.60 ml (2.69 mmol) of 2-cyanoethyl diisopropylchlorophosphoramidite,0.90 ml (5.2 mmol) dry Hunig's base and 0.14 ml (3.46 mmol) anhydrousmethanol (Aldrich) were reacted in 15 ml dry THF under argon withstirring. The reaction was allowed to proceed for over 20 hours andmonitored by TLC (a white-orange spot on staining with ninhydrin thatfollows the solvent front for a 50:50 mix of ethyl acetate-hexanes, orhas an R=0.12 in 5% ethyl acetate in hexanes). After this about 2.5 g ofsilica gel that had previously been neutralized with triethylamine wasthen added to the reaction mixture and the solid products were adsorbedonto the silica gel by removing the solvents by rotovaporization. Thedried down silica gel was collected into a Biotage FLASH sampleinjection (SIM) cartridge and eluted through a silica gel packed FLASH20S column with 7% ethyl acetate in hexanes on a Biotage FLASH 40chromatography system, pressure=15 psi. (Note: Before sample elution thecolumn was prepared by neutralizing the silica gel in it withapproximately 250 ml of a 15% triethylamine-methanol solution afterwhich it was rinsed with about 100 ml of the 7% ethyl acetate in hexanessolution). Fractions 6 to 10 were pooled and rotovaporized to give 0.372g (1.60 mmol) of product in 60% yield (to the initialchlorophosphoramidite).

Step B

0.3551 g (1.529 mmoles) of product from Step 1 was added to 4.0 ml (1.8mmol) of a 0.45 M 1H-tetrazole solution in acetonitrile (Fluka) in astoppered round-bottom flask under argon. Next, 3.7 ml (0.1515 g/ml=0.56g, 1.70 mmol) of a Combretastatin A4 (CA4) solution in dry methylenechloride was added slowly from a syringe dropwise to this solution underargon with stirring. The reaction was again monitored by TLC using a50:50 ethyl acetate-hexanes mixture, Rf of product=0.70 (visible by uv,turns brown by air oxidation of CA4 group and also develops orange withninhydrin) and allowed to react for about 33 hours.

Step C

After this 0.449 g of (˜2 mmol) m-choroperoxybenzoic acid (70-75%,Acros) was added to the reaction mixture and product formation wasmonitored by TLC as before. Almost complete conversion to the oxidizeddiester was evident in 10 minutes, but the reaction was run for 2 hours.TLC monitoring of the product showed a broad spot with an Rf of about0.30 in 50:50 ethyl acetate-hexanes. The product was purified by flashcolumn chromatography to give 0.250 g (0.540 mmol, 35% yield) of productshown pure by 1-H and 31-P NMR.

Step D

0.2122 g (0.4598 mmol) of pure product from Step 3 was dissolved in 10ml of an 80:20 methanol-methylene chloride solution to which was added0.15 ml (1.14 mmol, ˜2.5 equivalents) concentrated ammonia solution(28-30% ammonia, Acros) which was allowed to react for over 24 hours.The 3-aminopropionitrile by-product, ammonia and solvents were removedby rotovaporation and then vacuum pump. This gave the correct product inabout 90% yield verified by 1-H and 31-P NMR and greater than 90% inpurity by HPLC and capillary gel electrophoresis.

Using the procedures described herein or by modification of theprocedures described herein as known to one of ordinary skill in theart, the following additional compounds have been prepared:

2′-Disodium Phosphate-3′-Hydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene(ZSB-1A)

2′-Hydroxy-3′-Disodium Phosphate-3,4,41,5-tetramethoxy-(Z)-stilbene(ZSB-2A)

3′,5′-Dihydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-1B)

(0.27 g, 80%). ¹H NMR (300 MHz): 3.67(6H, s), 3.79(3H, s), 3.86(3H, s),4.96(2H, bs), 6.23(1H, d, J=12.28 Hz), 6.36(1H, d, J=12.2 Hz), 6.43(2H,s), 6.56(2H, s). ¹³C NMR (75.47 MHz): 55.92, 60.94, 61.14, 106.2,108.68, 129.19, 130.07, 132.29, 133.69, 134.02, 137.27, 148.62, 152.83.

3′,5′-Tetrasodium Phosphate-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-2B)

2′-Bromo-3′-Hydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-16)

(530 mg, 51.5%, solid). HNMR (ppm, δ): 6.83(1H, d, J=8.5), 6.70(1H, d,J=8.5 Hz), 6.56(2H, s), 6.42(2H, s), 3.94(3H, s), 3.88(3H, s), 3.65(6H,s) CNMR(ppm, δ): 153.13, 146.36, 143.54, 137.54, 132.43, 131.52, 131.10,129.14, 121.97, 110.61, 109.87106.44, 61.30, 56.83, 56.22

2′-Bromo-3′-Disodium Phosphate-3,4,4′,5-tetramethoxy-(Z)-stilbene(ZSB-17)

(120.2 mg, 0.2.3 mmol, 71.4%) ¹H-NMR(ppm, δ): 6.67(1H, q), 6.52(1H, d,J=11.9 Hz), 6.43(1H, d, J=12.4 Hz), 6.36(2H, s), 3.63(3H,s), 3.55(6H,s), 3.47(3H, s).

¹³-CNMR(ppm, δ, CDCl3): 152.32, 133.50, 130.96, 130.74, 129.96, 125.06,111.69, 106.71, 61.17, 56.11. P-NMR (ppm, δ): 1.06.

2′-hydroxy-3,3′,4,4′,5-pentamethoxy-(Z)stilbene (ZSB-18)

(1.49 g 4.3 mmol, 82.7%) HNMR(ppm, δ, CDCl3): 7.27(1H, d, J=12.3 Hz),7.08(1H, d, J=8.3 Hz), 7.03(1H, d, J=13.5 Hz), 6.75(2H, s), 6.53(1H, d,J=8.6 Hz), 3.92(6H, s), 3.90(6H, s), 3.88(3H, s). CNMR(ppm, δ, CDCl3):153.70, 152.05, 147.73, 137.83, 135.86, 134.36, 128.09, 123.12, 122.15,118.11, 104.43, 103.66, 61.39, 56.50, 56.27.

2′-Disodium Phosphate-3,3′,4,4′,5-pentamethoxy-(Z)stilbene (ZSB-19)

(97 mg, 0.21 mmol, 42%, solid) ¹H-NMR(ppm, δ, D2O): 7.25(1H, d, J=12.3Hz), 7.08(1H, d, J=8.3 Hz), 6.93(1H, d, J=13.6 Hz), 6.83 (1H, d, J=8.6Hz), 6.71(2H, s). PNMR (ppm, δ, D2O): 2.97

3′-hydroxy-3,4,4′,5,5′-pentamethoxy-(Z)stilbene (ZSB-20)

(560 mg 1.62 mmole, 62.3%). ¹H-NMR (ppm, δ, CDCl3): 6.88(2H, s),6.80(1H, s), 6.72(2H, s), 6.63(1H, s), 3.92(12H, s), 3.86(3H,s)C-NMR(ppm, δ, CDCl3): 153.79, 152.84, 149.83, 135.70, 133.83, 133.37,128.70, 128.30, 106.47, 103.84, 102.86, 61.40, 60.83. 56.52, 56.27.

3′-Hydroxy-2′,3,4,4′,5-pentamethoxy-(Z)-stilbene (ZSB-27A)

(251.5 mg, 0.726 mmol, 72.6%) ¹H-NMR (300 MHz, CDCl3): δ 6.73 (d, J=8.7Hz, 1H), 6.58 (d. J=12.3 Hz, 1H), 6.52-6.44 (m, 4H), 5.80 (s, 1H), 3.85,3.79 (s,s, 9H), 3.63(s, 6H).CDCl₃):δ 153.1, 147.5, 145.8, 138.9, 137.4,133.0, 130.4, 125.4, 124.2, 120.9, 120.6, 106.8, 106.4, 61.3, 61.2,56.7, 56.6, 56.2.

3′-Disodium Phosphate-3,3′,4,4′,5-pentamethoxy-(Z)-stilbene (ZSB 27B)

(117.8 mg, 0.25 mmol, 73.7%, solid). ¹H-NMR (300 MHz, D₂O): δ 6.70 (d,J=8.4 Hz, 1H), 6.55 (d,d J=12.1 Hz, 5.5 Hz, 1H), 3.70, 3.56, 3.48,(s,s,s, 15H). ¹³C-NMR (300 MHz, D₂O): δ 153.2, 152.3, 150.9, 137.1,136.9, 136.0, 134.0, 126.5, 124.2, 124.1, 108.3, 106.7, 61.2, 61.1,56.2, 56.1. ³¹P-NMR (300 MHz, D₂O): δ 1.43

4′-hydroxy-2′-Iodo-3,4,5,5′-tetramethoxy-(Z)-stilbene (ZSB-29A)

(0.72 g, 1.5 mmol, 88%, oil). ¹H-NMR (CDCl₃, 300 MHz): δ 3.67 (s, 3H);3.73 (s, 6H); 3.84 (s, 3H); 6.09 (s, 1H); 6.39 (d, J=12.1, 1H); 6.47 (d,J=12.1, 1H); 6.53 (s, 2H); 6.74 (d, J=1.6, 1H); 7.30 (d, J=1.6, 1H).

4′-Disodium Phosphate-2′-Iodo-3,4,5,5′-tetramethoxy-(Z)-stilbene(ZSB-29B)

2′,5′-Dihydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-33A)

2′,5′-Tetrasodium Diphosphate-3,4,4′,5-tetramethoxy-(Z)-stilbene(ZSB-33B)

2′,3′-Dihydroxy-3,4,5-trimethoxy-(Z)-stilbene (ZSB-36A)

(0.5 g, 1.65 mmol, 43.3%) ¹H-NMR (CDCl₃, 300 MHz): δ 3.63 (6H,s,2*OCH₃);3.83 (3H, s, OCH₃), 5.10 (1H,s, OH), 5.50 (1H,s,OH), 6.47(2H,s,H-2,H-6), 6.53 (1H,d,J=12.04 Hz, —CH═CH—), 6.60 (1H,d,J=12.06 Hz,—CH═CH—), 6.92 (3H,m,H-4′,H-5′,H-6′).

2′,3′-Diphosphate-3,4,5-trimethoxy-(Z)-stilbene (ZSB-36B)

(0.036 g, 0.071 mmol, 59.1%, solid) ¹H-NMR (300 MHz, D₂O): δ 3.69(6H,s,2*OCH₃); 3.77 (3H, s, OCH₃), 6.66 (2H,s, H-2,H-6), 6.73(1H,d,J=12.02 Hz, —CH═CH—), 6.86 (1H,d,J=12.21 Hz, —CH═CH—), 6.98(2H,m,H-5′,H-6′), 7.24 (20H, m, 4*C6H6), 7.36 (1H,d,J=7.14 Hz, H-4′)PNMR (300 Mhz, D2O) 6-3.27, −3.88.

3′,4′ Dihydroxy-3,4,5-trimethoxy-(Z)-stilbene (ZSB-37A)

3′,4′ Diphosphate-3,4,5-trimethoxy-(Z)-stilbene (ZSB-37B)

¹H-NMR (ppm, δ, D2O): 6.77 (1H,s); 6.73 (2H, q); 6.52 (2H, s); 6.46(1H,d,J=12.1 Hz), 6.41 (1H,d, J=12.1, 1H), 3.88 (3H,s), 3.72 (6H,s).PNMR (ppm, δ, D2O): −3.69.

4′-hydroxy-3,4,5-trimethoxy-(Z)-stilbene (ZSB-40A)

(ZSB-40B) 4′-phosphate-3,4,5-trimethoxy-(Z)-stilbene

(85 mg, 0.20 mmol, 30.0%). ¹H-NMR (ppm, δ): 7.13 (1H,d, J=8.3 Hz),7.00(1H,d, J=8.3 Hz), 6.59 (2H,s), 6.56 (1H,d,J=13.00), 6.45(1H,d,J=12.1Hz), 3.67(3H,s).

¹³C-NMR(ppm, δ): 153.00, 133.50, 130.00, 129.80, 129.76, 125.00, 120.05,119.50, 108.00, 106.44, 61.00, 56.01. PNMR (ppm, δ): 0.10.

2′-Fluoro-3′-Hydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-41A)

(126 mg, 0.37 mmol, 74.8%). ¹H-NMR (300 MHz, CDCl₃): δ 6.79 (t, J=8.4Hz, 1H), 6.57-6.50 (m, 5H), 3.86(s, 3H), 3.85 (s, 3H), 3.80, (s, 6H).¹³C-NMR (300 MHz, CDCl₃): δ 153.2, 150.6, 147.9, 147.4, 137.6, 134.4,134.2, 132.8, 131.6, 122.33, 122.29, 120.27, 1 119.25, 119.08, 106.3,61.29, 56.78, 56.25

(ZSB-41B) 2′-Fluoro-3′-DisodiumPhosphate-3,4,4′,5-tetramethoxy-(Z)-stilbene

2′-Hydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-46A)

(620 mg, 55%, solid). HNMR(ppm, δ, CDCl3): 7.43(1H, J=8.6 Hz), 7.20(1H,d, J=16.3 Hz), 6.94(1H, d, J=16.3 Hz), 6.74(2H, s), 6.55(1H, d, J=8.3Hz), 3.92(6H, s), 3.88(3H, s), 3.81(3H, s).

2′-Disodium Phosphate-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-46B)

HNMR(ppm, δ, D2O): 7.49(1H,d0, 7.36 (1H,d), 6.94 (4H,m), 6.57 (1H,d),3.77(6H,s), 3.71(3H,s), 3.65 (3H,s). PNMR(ppm, δ, D2O): 1.26.

3,5-Dinitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-13)

3′,5′-Diamine-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-14)

¹H NMR (CDCl₃, 360 MHz) δ 6.56 (s, 2H), 6.41 (d, 1H, J=12.2 Hz), 6.34(d,1H, J=12.2 Hz), 6.14 (s, 2H), 3.82 (s, 3H), 3.72 (s, 3H), 3.70 (s,6H).

3′,5′-Diserinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-15)

2′-Nitroso-3′hydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-28A)

(80 mg, 0.23 mmole, 65%, solid) ¹H NMR: 3.64 (s, 6H, 2×OCH₃); 3.81 (s,3H, OCH₃); 3.92 (s, 3H, OCH₃); 6.30 (s, 2H, aryl); 6.59 (d, J=12, 1H);6.68 (d, J=12, 1H); 6.78 (d, J=8.4, 1H); 6.92 (d, J=8.4, 1H).

2′-Nitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-39A)

(5.29 g, 81% yield) ¹H NMR (CDCl₃, 300 MHz) 67.59 (d,1H, J=2.7 Hz), 7.24(d,1H, J=8.8 Hz), 7.01 (dd, 1H, J=8.7, 2.7 Hz), 6.80 (d,1H, J=11.9 Hz),6.62 (d,1H, J=12.0 Hz), 6.28 (s, 2H), 3.93 (s, 3H), 3.86 (s, 3H), 3.62(s,6H).

2′-Amino-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-39B)

(0.817 g, 38%). ¹H NMR (CDCl₃, 360 MHz) 67.03 (d, 1H, J=8.4 Hz), 6.51(s,2H), 6.49 (d,1H, J=11.6 Hz), 6.42 (d, 1H, J=12 Hz), 6.30 (dd, 1H,J=8.4, 2.5 Hz), 6.25 (d, 1H, J=2.4 Hz), 3.80 (s, 3H), 3.75 (s, 3H), 3.64(s, 6H), 1.55 (br, 1H).

2′-Serinamide-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-45)

(0.817 g, 38%). ¹H NMR (CDCl₃, 360 MHz) 67.03 (d, 1H, J=8.4 Hz), 6.51(s,2H), 6.49 (d,1H, J=11.6 Hz), 6.42 (d, 1H, J=12 Hz), 6.30 (dd, 1H,J=8.4, 2.5 Hz), 6.25 (d, 1H, J=2.4 Hz), 3.80 (s, 3H), 3.75 (s, 3H), 3.64(s, 6H), 1.55 (br, 1H).

3′-Hydroxy, 5′-Nitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-43)

¹H NMR (CDCl₃, 300 MHz): 3.72 (s, 6H), 3.85 (s, 3H), 3.94 (s, 3H), 6.42(d, 1H, J=12.1 Hz), 6.47 (s, 2H), 6.61 (d, 1H, J=12.1), 7.16 (d, 1H,J=2.0 Hz), 7.39 (d, 1H, J=2.0 Hz).

¹³C NMR (CDCl₃, 75 MHz): 56.04, 61.00, 62.54, 106.06, 117.286, 120.73,126.81, 131.34, 132.38, 133.92, 137.92, 139.80, 142.64, 150.15, 153.18.

3′-Hydroxy, 5′-Amino-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-44)

(60 mg, 15%, oil). ¹H NMR (CDCl₃, 300 MHz): 3.71 (s, 6H), 3.78 (s, 3H),3.84 (s, 3H), 6.27 (d, 1H, J=1.9), 6.35 (d, 1H, J=1.9), 6.41 (d, 2H,J=1.5), 6.54 (s, 2H).

2′-Amino-3′hydroxy-3,4,4′,5-tetramethoxy-(Z)-stilbene (ZSB-48)

(90.5 mg, 82%, oil) ¹H NMR (CDCl₃, 300 MHz): 3.64 (s, 6H), 3.81 (s, 3H),3.84 (s, 3H), 6.32 (d, 1H, J=8.4), 6.45 (d, 1H, J=12.1), 6.51 (d, 1H,J=12.0), 6.51 (s, 2H), 6.68 (d, 1H, J=8.4). ¹³C NMR (CDCl₃, 75 MHz):55.7, 56.1, 60.8, 101.1, 105.9, 117.6, 120.1, 125.7, 130.9, 132.1,132.2, 132.6, 137.2, 145.7, 152.7.

CA4P Diethyl Ester (Oxi-com 157)

CA4P Dimethyl Ester (Oxi-com 184)

CA4P Cyclohexane Ester, Ammonium Salt (Oxi-com 191)

CA4P 4-Chlorobenzyl Ester, Ammonium Salt (Oxi-com-192)

CA4P n-Octyl Ester, Ammonium Salt (Oxi-com 210)

CA4P Trifluoroethane Ester, Ammonium Salt (Oxi-com 211)

Example 7 Biological Activity

The pharmacological properties of the compounds of this invention may beconfirmed by a number of pharmacological assays. The exemplifiedpharmacological assays have been carried out with several of thecompounds of the present invention.

A. MTT Cytotoxicity Assay

Exponentially growing were treated with the following compounds for 1hour and 5 days. Insoluble compounds were formulated in a small amount(0.3%) of DMSO for biological evaluation. Cell viability was determinedby the calorimetric MTT assay using3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromideaccording to well-established procedures (see Berridge, et al. (1996)for a general protocol of this type of assay). The results are shown inTable 2.

TABLE 2 Compound IC50 @ 1 h (uM) IC50 @ 5 days (uM) ZSB-2B 25 1 ZSB-3B2.4 0.0043 ZSB-16 2 0.0067 ZSB-26A 3.07 0.016 ZSB-27 16 0.016 ZSB-39 20.008 ZSB-41A 8 0.015 ZSB-43 8 1.3 ZSB-45 10 0.05 ZSB-46A 43 0.068Oxi-com183 8 0.26 Oxi-com191 35 0.13 Oxi-com209 38 0.07 Oxi-com210 250.07

B. Vascular Shutdown Assay

The vascular effects of the following compounds were assessed intumor-bearing mice using a fluorescent-bead assay. A MHEC-5Themangioendothelioma tumor model was established by subcutaneousinjection of 0.5×10⁶ cultured MHEC5-T cells into the right flank of FoxChase CB-17 SCID mice and allowed to grow to a size of 300 mm³ beforei.p. injection with a single dose of saline control or compound. At 24hours post-treatment, mice were i.v. injected with 0.25 ml of dilutedFluoSphere beads (1:6 in physiological saline) in the tail vein, andsacrificed after 3 minutes. Tumor cryosections at a thickness of 8 umwere directly examined using quantitative fluorescent microscopy. Forquantification, image analysis of 3 sections from three tumor treated ineach group were examined and vascular shutdown was expressed a vesselarea per tissue area (mm²) in percentage of the control. The results areshown in Table 3.

TABLE 3 % Blood Flow Shutdown Compound (100 mg/kg) ZSB-2B 65 ZSB-21 46ZSB-27B 41 ZSB-29B 43 ZSB-33B 51 ZSB-39B 50 ZSB-45 43 Oxi-com192 90Oxi-com210 89

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

It is also to be understood that the drawings are not necessarily drawnto scale, but that they are merely conceptual in nature.

The following references are incorporated herein by reference in theirentirety:

-   Aleksandrzak, K., et al., (1998). “Antimitotic Activity of Diaryl    Compounds with Structural Features Resembling Combretastatin A-4.”    Anti-Cancer Drugs 9: 545-550.-   Bedford, S. B., et al., (1996). “Synthesis of Water-Soluble Prodrugs    of the Cytotoxic Agent Combretastatin A-4.” Bioorganic and Medicinal    Chemistry Letters 6(2): 157-160.-   Berridge M. V., et al., (1996). “The biochemical and cellular basis    of cell proliferation assays the use Tetrazolium salts” Biochemica    4: 15-19.-   Brown, M. L., et al., (2000). “Comparative Molecular Field Analysis    of Colchicine Inhibition and Tubulin Polymerization for    Combretastatins Binding to the Colchicine Binding Site on Beta    Tubulin.” Bioorganic and Medicinal Chemistry 8: 1433-1441.-   Chen, Z., et al., (2000). “Preparation of New Anti-Tubulin Ligands    through a Dual-Mode, Addition-Elimination Reaction to a    Bromo-Substituted    □-Unsaturated Sulfoxide.” Journal of Organic Chemistry 65(25):    8811-8815.-   Cushman, M., et al., (1992). “Synthesis and Evaluation of Analogues    of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethane as    Potential Cytotoxic and Antimitotic Agents.” Journal of Medicinal    Chemistry 35(12): 2293-2306.-   Dark, et al., (1997). “Combretastatin A4, an Agent that Displays    Potent and Selective Toxicity toward Tumor Vasculature.” Anticancer    Research 57: 1829-1834.-   del Rey, B., et al., (1999). “Leishmanicidal Activity of    Combretastatin Analogues and Heteroanalogues.” Bioorganic and    Medicinal Chemistry Letters 9: 2711-2714.-   Deshpande, V. H., et al., (1992). “Synthesis of Combretastatin D-2.”    Tetrahedron Letters 33(29): 4213-4216.-   El-Zayet, A. A. E., et al., (1993). “In vitro Evaluation of the    Antineoplastic Activity of Combretastatin A-4, a natural product    from Combretum caffrum.” Anticancer Drugs 4: 19-25.-   Galbraith, S. M., et al., (2001). “Effects of Combretastatin A4    Phosphate on Endothelial Cell Morphology In Vitro and Relationship    to Tumour Vascular Targeting Activity in VIvo.” Anticancer Research    21: 93-102.-   Griggs, J., et al., (2001). “Potent Anti-Metastic Activity of    Combretastatin-A4.” International Journal of Oncology 19: 821-825.-   Gwaltney, S. L., et al., (2000). “Novel Sulfonate Analogues of    Combretastatin A-4: Potent Antimitotic Agents.” Bioorganic and    Medicinal Chemistry Letters 11: 871-874.-   Hatanaka, T., et al., (1998). “Novel B-ring Modified Combretastatin    Analogues: Syntheses and Antineoplastic Activity.” Bioorganic and    Medicinal Chemistry Letters 8: 3371-3374.-   Hori, K., et al., (2001). “Stoppage of Blood Flow in    3-methylcholanthrene-induced Autochthonous Primary Tumor due to a    Novel Combretastatin A-4 derivative, AC7700, and its Antitumor    Effect.” Medical Science Monitor 7(2): 26-33.-   Hori, K., et al., (1999). “Antitumor Effects due to Irreversible    Stoppage of Tumor Tissue Blood Flow: Evaluation of a Novel    Combretastatin A-4 Derivative, AC7700.” Jpn. J. Cancer Research 90:    1026-1038.-   Iyer, S., et al., (1998). “Induction of Apoptosis in Proliferating    Human Endothelial Cells by the Tumor Specific Antiangiogenesis Agent    Combretastatin A-4.” Cancer Research 58: 4510-4514.-   Katsuyoshi, H., et al., (1999). “Antitumor Effects due to    Irreversible Storage of Evaluation of a Novel Combretastatin A-4    Derivatives, AC7700.” Jpn. J. Cancer Research 90: 1026-1039.-   Maya, A. B. S., et al., (2000). “Design, Synthesis, and Cytotoxic    Activities of Naphthyl Analogues of Combretastin A-4.” Bioorganic    and Medicinal Chemistry Letters 10: 2549-2551.-   McGown A. T., et al., (1989). “Structural and Biochemical    Comparision of the Anti-mitotic Agents Colchicine, Combretastin A-4    and Amphethinile.” Anti-cancer Drug Design 3: 249-254.-   McGown A. T., et al., (1990). “Differential Cytotoxicity of    Combretastatins A1 and A4 in Two Daunorubicin-Resident P388 Cell    Lines.” Cancer Chemotherapy and Pharmacology 26: 79-81.-   Medarde, M., et al., (1998). “Synthesis and Antineoplastic Activity    of Combretastatin Analogues: Heterocombretastatins.” Eur J Nucl Med    33: 71-77.-   Medarde, M., et al., (1999). “Synthesis and Pharmacological Activity    of Diarylindole Derivatives. Cytotoxic Agents Based on    Combretastatins.” Bioorganic and Medicinal Chemistry Letters 9:    2303.-   Medarde, M., et al., (1995). “Synthesis and Pharmacological Activity    of Combretastatin Analogues. Naphthylcombretastatin and Related    Compounds.” Bioorganic and Medicinal Chemistry Letters 5(3):    229-232.-   Nihei, Y., et al., (1999). “A Novel Combretastatin A-4 Derivative AC    7700, Shows Marked Antitumor Activity against Advanced Solid Tumors    and Orthotopically Transplant Tumors.” Jpn. J. Cancer Research 90:    1016-1025.-   Ohsumi, K., et al., (1998). “Syntheses and Antitumor Activity of Cis    Restricted Combretastatins: 5 Membered Heterocyclic Analogues.”    Bioorganic and Medicinal Chemistry Letters 8: 3153-3158.-   Pedley, R. B., et al., (2001). “Eradication of Colorectal Xenografts    by Combined Combretastatin A-4 3—O— Phosphate.” Cancer Research 61:    4716-4722.-   Pettit, George R. Combretastatin A-4 Prodrug-Anti-Tumor    Chemotherapy. U.S. Pat. No. 5,561,122.-   Pettit, George R. Cell Growth Inhibitory Macrocyclic Lactones    Denominated-   Combretastatin D-1 and D-2. U.S. Pat. No. 4,940,726.-   Pettit, George R., Sheo B. Singh. Combretastatin A-4—Tubulin    Polymerization Inhibitor; Antitumor Agent. U.S. Pat. No. 4,996,237.-   Pettit, G. R., et al., (1999). “Antineoplastic Agents. 410.    Asymmetric Hydroxylation of trans-Combretastatin A-4.” Journal of    Medicinal Chemistry 42: 1459-1465.-   Pettit, G. R., et al., (1998). “Antineoplastic Agents. 379.    Synthesis of Phenstatin Phosphate.” Journal of Medicinal Chemistry    41: 1688-1695.-   Pettit, G. R., et al., (1995). “Antiangioplastic agents 322.    Synthesis of combretastiatin A-4 prodrugs.” Anti-cancer Drug Design    10: 299-309.-   Pettit, G. R., et al., (1982). “Isolation and Structure of    Combretastatin.” Canadian Journal of Chemistry 60: 1374-1376.-   Pettit, G. R., and John W. Lippert (2000). “Antineoplastic    Agents 429. Syntheses of the Combretasatin A-1 and Combretastatin    B-1 prodrug.” Anti-cancer Drug Design 15: 203-216.-   Pettit, G. R., Sheo Bux Singh (1987). “Isolation, Structure, and    Synthesis of Combretasatin A-2, A-3, and B-2.” Canadian Journal of    Chemistry 65: 2390.-   Pinney, K. G., et al., (2000). “Synthesis and Biological Evaluation    of Aryl Azide Derivatives of Combretastatin A-4 as Molecular Probes    for Tubulin.” Bioorganic and Medicinal Chemistry 8: 2417-2425.-   Rey, B. d., et al., (1999). “Leishmanicidal Activity of    Combretastatin Analogues and Heteroanalogues.” bioorganic and    Medicinal Chemistry Letters 9: 2711-2714.-   Russell, G., et al., (1995). “Inhibition of [H] Mebendazole Binding    to Tubulin by Structurally Diverse Microtubul Inhibitors which    Interact at the Colchicine Binding Site.” Biochemistry and Molecular    Biology International 35(6): 1153-1159.-   Sackett, D. L. (1993). “Podophyllotoxin, Steganacin and    Combretastatin: Natural Productsl that Bind at the Colchicine Site    of Tubulin.” Pharmarc. Ther. 59: 163-228.-   Schwikkard, S., et al., (2000). “Bioactive Compounds from Combretum    erythrophyllum.” Journal of Natural Products 63: 457-460.-   Sello, G., et al., (1996). “Using a Canonical Matching to Measure    the Similarity Between Molecules: The Taxol and the Combretastatin    A1 Case.” Advances in Molecular Similarity 1(243-266).-   Shirai, R., et al., (1998). “Asymmetric Synthesis of Antimitotic    Combretadioxolane with Potent Antitumor Activity Against Multidrug    Resistant Cells.” Bioorganic and Medicinal Chemistry Letters 8:    1997-2000.-   Shirai, R., et al., (1997). “Synthesis of Conformationary Restricted    Combretastatins.” Heterocycles 46: 145-148.-   Springer Matthew L., et al., (2000). Angiogensis monitored by    perfusion with a space-filling microbead suspension. Molecular    Therapy 1: 82-87.-   Tan, L. P., et al., (1975). “Effects of Indole Alkaloids and Related    Compounds on the Properties of Brain Microtubular Protein.” Biochem.    Sec. Trans. 3(1): 121-124.-   Tozer, G. M., et al., (2001). “Mechanisms Associated with Tumor    Vascular Shut-Down Induced by Combretastatin A-4 Phosphate:    Intravital Microscopy and Measurement of Vascular Permeability.”    Cancer Research 61: 6413-6422.-   Watts, M. E., et al., (1997). “Effects of Novel and Conventional    Anti-Cancer Agents on Human Endothelial Permeability: Influence of    Tumour Secreted Factors.” Anticancer Research 17: 71-76.-   Zhao, S., et al., (1999). “Positron Emission Tomography of Murine    Liver Metastases and the Effects of Treatment by Combretastatin    A-4.” Eur J Nucl Med 26: 231-238.

1. A method for treating a vascular proliferative disorder in a hostcomprising administering to a host an effective amount of the compoundof the formula I:

or a pharmaceutically acceptable salt or hydrate thereof wherein: R₁,R₄, and R₅ are each independently H, OH, lower alkoxy, NH₂, NO₂, N₃,NHR₆, halogen, or a phosphate ester salt moiety of the general formula(—OP(O)O⁻M⁺)₂,(—OP(O)(OR₉)(O⁻M⁺), or —OPO₃R₇R₈; R₂ is H, OH, loweralkoxy, NH₂, NO₂, NHR₆, or a phosphate ester salt moiety of the generalformula (—O—P(O)(O⁻M⁺)₂, (—OP(O)(OR₉)(O⁻M⁺), or —OPO₃R₇R₈, wherein NH₂or OH may form a ring with R₁; R₃ is H, lower alkoxy, or a phosphateester salt moiety of the general formula (—OP(O)(O⁻M⁺)₂,(—OP(O)(OR₉)(O⁻M⁺), or —OPO₃R₇R₈; R₆ is acylamino group; R₇ is ammoniumsalt (NH₄ ⁺); R₈ is lower alkyl, cycloalkyl, or aryl; R₉ is alkyl,branched alkyl, benzyl, or aryl; and M is a metal cation or salt,provided that when R₁, R₄, and R₅ are H, R₂ is H, lower alkoxy, NH₂,NO₂, NHR₆, or a phosphate ester salt moiety of the general formula(—OP(O)(OR₉)(O⁻M⁺) or —OPO₃R₇R₈; and provided that when R₁, R₂, and R₅are H, R₄ is H, lower alkoxy, NH₂, NO₂, NHR₆, or a phosphate ester saltmoiety of the general formula (—OP(O)(OR₉)(O⁻M⁺) or —OPO₃R₇R₈, whereinsaid vascular proliferative disorder is selected from wet maculardegeneration, diabetic retinopathy, retinopathy of prematurity,restenosis, and a cancer selected from leukemia, lung, colon, thyroid,melanoma, ovarian, renal, prostate, and breast cancer.
 2. The method ofclaim 1, wherein said host is a mammal.
 3. The method of claim 2,wherein said mammal is a human.
 4. The method of claim 1, wherein saidcompound is administered systemically.
 5. The method of claim 1, whereinsaid compound is administered orally.
 6. The method of claim 1, whereinsaid compound is administered intravenously.
 7. The method of claim 1,wherein said compound is administered topically.
 8. The method of claim1, wherein said compound is administered along with a carrier.